Maternal diet influences fecundity in a freshwater turtle undergoing population decline

Maternal diet affects reproductive output in female freshwater turtles (Emydura macquarii). At wetlands where filamentous green algae are scarce and turtles are functionally carnivorous, females produce smaller clutches. Reduced clutch size associated with food availability may have implications for declining freshwater turtle species.


Introduction
When food is abundant, available, and not limiting, an animal can theoretically allocate sufficient energy to every physiological and behavioural process necessary to maximize fitness (Congdon et al., 1982;Dunham et al., 1989).However, when food is limited, maximizing fitness requires trade-offs in energy allocation among these processes.When unable to gather food sufficient to maintain all of these processes, adult animals may (i) reduce their allocation to growth and sacrifice future (size-dependent) fecundity in order to meet immediate needs, (ii) cease or reduce reproduction until conditions and food availability improves and (iii) reduce the allocation to maintenance and other allocations with a direct bearing on survival such as immune responses.In long-lived species, there are limits to how much of the allocation of available energy directed to processes affecting immediate survival can be reduced (Stoeckmann and Garton, 2001;Beaupre, 2007).A trade-off arises between fitness gain of reproduction in the immediate or short term, and loss of opportunity to capitalize on the fitness benefits of reproduction in future years, should the mother perish (Stoeckmann and Garton, 2001;Beaupre, 2007).The outcome of these trade-offs can explain the evolution of species that fall on the spectrum from semelparity to extreme iteroparity (Bonnet, 2011), where most turtles arguably reside.
Availability of food, moderated by stored resources, determines the amount of energy a female animal can allocate to reproduction at the time of a reproductive event (Congdon, 1989;Bernardo, 1996;Mousseau and Fox, 1998).Most research on the effects of food limitation on animals specifically focuses on the consequences of energy limitation.However, food limitations (i.e.reductions in the amount eaten or changes to the composition of the diet) can also reduce the total nutritional content available to an animal (Ramsay and Houston, 1997).Based on the availability of energy or essential nutrients, females may vary the frequency of reproductive events per year (or over longer time periods), the number of offspring produced and/or the size of offspring (Congdon, 1989;James and Whitford, 1994;Shine and Madsen, 1997;Warner et al., 2007;Van Dyke and Griffith, 2018).Theory predicts that a trade-off also occurs between the number and size of offspring produced, with females either producing many small offspring or few large offspring (Smith and Fretwell, 1974;Rollinson and Hutchings, 2013;Stahlschmidt and Adamo, 2015).
Food type (i.e.plant, animal or both) eaten by females during reproduction or during a prior accumulation period may also affect the availability of nutrients to offspring during development.Carnivory is typically associated with higher consumption of protein and calcium (Clark and Gibbons, 1969;Sánchez-Vázquez et al., 1999), whereas herbivory is associated with higher consumption of fat, carbohydrates, potassium and sodium (King, 1996;Sánchez-Vázquez et al., 1999;Sterner and Elser, 2002).Likewise, food availability may also affect the allocation of essential and non-essential nutrients (especially amino acids and fatty acids) to offspring (Guisande et al., 1999).Under food limitation, essential nutrients (those which cannot be synthesized from other nutrients and must be present in the diet), may be reduced or absent (Wu, 2009).In contrast, non-essential nutrients can be synthesized by the females from their diet and may be less affected (Wu, 2009).In oviparous species, the egg contains all of the nutrients required by the embryo for successful development (Congdon et al., 1983).Mothers allocate available nutrients directly from food consumed; from storage in the liver or fat deposits; and from storage of egg yolk during vitellogenesis, protein to the albumen layer and calcium to the eggshell (Van Dyke and Griffith, 2018).
We determined how reproduction by female Murray River short-necked turtles (Emydura macquarii) is affected by turtle diet composition (i.e.relative abundances of dietary plant and animal species) by comparing differences in maternal diet and allocation of resources to reproduction among females at four wetlands in north-central Victoria, Australia.We used E. macquarii as a model species because it is a generalist consumer that is able to change its diet as food availability changes (Chessman, 1986;Spencer et al., 2014;Petrov et al., 2020).We have previously found that E. macquarii diets differ among the four abovementioned sites and that the sites vary in the availability and accessibility of prey (Petrov et al., 2018;Petrov et al., 2020).Emydura macquarii is also important as a model species because they are listed as vulnerable in Victoria (Van Dyke et al., 2018) and have declined by as much as 67% across the Murray River catchment (Chessman, 2011;Van Dyke et al., 2019).Populations of E. macquarii in the Murray River catchment are heavily biased towards older individuals, particularly females, with juveniles being uncommon.A lack of juveniles suggests low rates of recruitment, potentially owing to high rates of nest predation by the European red fox Vulpes vulpes (Thompson, 1993;Van Dyke et al., 2019).Additionally, the availability of food (i.e.relative abundances of dietary plant and animal species) and its effects on reproduction has not previously been studied as a factor in their decline.Specifically, we tested for differences in total clutch mass, individual egg mass and hatching success across the four wetlands.Because maternal diet constrains the nutrients available to be allocated to each egg (Congdon et al., 1983;Finkler and Claussen, 1997), we also compared the bulk composition of eggs from females from each wetland.Specifically, we determined the amount of water, total protein, total lipid and energy allocated to each egg.We also repeated our prior stable isotope comparison (Petrov et al., 2020) to verify that the turtles in these wetlands still exhibited the same dietary differences.We predicted that females from Longmore Lagoon (low filamentous algae availability, high carnivory, empty stomachs) would exhibit reduced reproductive output, such that clutch mass and individual egg mass are reduced, compared to females from Safes Lagoon (high filamentous algae availability, high herbivory, no empty stomachs), and that females from Cockatoo Lagoon and Gunbower Creek (intermediate food availability) would exhibit some intermediate values for the above parameters (Petrov et al., 2020).Lastly, we compared hatching success across sites to determine whether any differences we detected in egg composition were associated with developmental success.

Study sites
This study was conducted at four wetlands adjacent to the Murray River between Cohuna and Gunbower, Victoria, Australia: the wide-bodied oxbow lakes Cockatoo Lagoon (35.919,144.360),144.393)and Safes Lagoon (−35.687, 144.156), which are connected to Gunbower Creek (−35.861, 144.331) by a regulated network of channels and pipes (Petrov et al., 2018).Our previous research found E. macquarii at Safes Lagoon consume large amounts of filamentous green algae, whereas E. macquarii at Longmore Lagoon are more carnivorous (Petrov et al., 2018;Petrov et al., 2020).At Longmore Lagoon, more turtles had empty stomachs and female turtles had lower body condition (Petrov et al., 2020), both potential consequences of reduced food availability.At Cockatoo Lagoon and Gunbower Creek, E. macquarii are omnivorous (Petrov et al., 2018).These dietary differences were verified using both stomach contents and stable isotope analyses and were driven by differences in local food availability (Petrov et al., 2018;Petrov et al., 2020).Although E. macquarii is likely capable of moving between at least some of the four wetlands, the isotopic differences we previously reported (Petrov et al., 2020) suggest either that such movements are rare or that turtles forage primarily in only one area for prolonged periods of time.

Turtle sampling and egg collection
Emydura macquarii were trapped during Austral Spring between 30 October 2016 and 6 November 2016, using baited cathedral traps.Traps were set at least 5 m apart and were checked every 10-14 hr (Petrov et al., 2020).Trapping continued until ∼10 gravid female E. macquarii, as determined by palpation, were sampled from each site.Gravid females that were not ready to lay, as indicated by the presence of soft eggs relatively anterior in the body cavity, were released with non-gravid females.Gravid females were weighed (to the nearest grams, using scales up to 5 kg) and measured using large (up to 102 cm) callipers (carapace and plastron length and width, to the nearest millimeter).
The gravid females were held on-site in water-filled tubs and injected intramuscularly with 20 mg/kg of oxytocin to induce oviposition (Ewert and Legler, 1978); turtles were reinjected every 48 hr until no eggs were felt via palpation, but only one injection was necessary for the majority of turtles.
The number of eggs laid by each female and the weight (to the nearest grams measured with digital scales), length and width (to the nearest millimeters, measured with vernier callipers) of each egg were recorded (see Supplementary Material, Tables S6-S9).The first and last eggs of each clutch were frozen and stored at −20 • C for isotopic and compositional analysis.After oviposition, females were reweighed.Claw clippings were sampled from each of the toes of the left hind leg of each turtle for stable isotope analysis (Petrov et al., 2020).Claw clippings reflect the isotopic composition of the food eaten over the past ∼12 months (Seminoff et al., 2007).Claw samples were frozen and stored at −20 • C for stable isotope analysis.
The remaining eggs in each clutch were labelled with a pencil to identify site, maternal identity and laying order within the clutch.The eggs were placed in moist vermiculite and transported to Western Sydney University for incubation.Each clutch was divided in half into one of two incubation temperatures, with 'even numbered' eggs incubated at 26 • C and 'odd numbered eggs' incubated at 30 • C.These incubation temperatures were used for another study concurrent with this project.The two treatments were incubated in separate incubators.Within both incubators, eggs were grouped by clutch and placed in covered (but unsealed) plastic tubs filled with a 1:1 mixture, as determined by weight, of vermiculite and deionized water.The mass of each tub was measured weekly, and any mass lost from evaporation of water was replaced with water from a spray bottle to maintain the water content of the vermiculite in each container (Packard et al., 1987).Tubs were randomly rotated around the incubator to account for shelf-specific variations in temperature.Eggs that did not develop or developed fungi were removed from the clutch and frozen at −20 • C. We recorded the duration of incubation in days.Upon hatching, the hatchlings were weighed (to the nearest grams with a digital scale) and measured (carapace and plastron length and width, to the nearest millimeters, with vernier callipers) for a concurrent study.The number of successful hatchings was recorded.This work was conducted under Western Sydney University Animal Ethics Committee Animal Research Authority A11794.This work was conducted under DEWLP permit 10 008 041 and DEPI permit RP1225.

Compositional analysis of eggs
All frozen eggs samples and maternal claw clippings were freeze dried at −40 • C to asymptotic mass using an Edwards Modulyo Freeze Dryer (Burgess Hill, United Kingdom).Eggs (including the shell) and claw clippings were homogenized to powder using a Retsch 400MM ball mill (Haan, Germany) and stored in a desiccator until further analysis.We estimated the moisture, protein, lipid and energy contents of each homogenized egg (i.e. two eggs per clutch, n = 62).We determined the water content (g) of each egg by subtracting the dry mass of the egg from the initial wet egg mass, when it was first laid.Total protein content was determined by measuring  (2014).As per the stable isotope analyses below, 1 mg of egg homogenate was placed into tin capsules and packaged into 96-well microplates prior to analysis.The nitrogen content (%) of each sample was multiplied by 6.25, following the Dumas method (Jung et al., 2003) to estimate the protein content (%) of each sample.Percentage protein was then multiplied by the dry mass of the sample to determine total bulk protein mass (g).Total lipid was determined by a twostep extraction and separation process.Lipids were extracted from a subsample of homogenate (∼0.5 mg) via further homogenization in a chloroform-methanol-water (1:1:1) mix using a Thomas Scientific PYREX dounce homogenizer tissue grinder.The subsample homogenate was filtered through a Büchner funnel and transferred to a 50-ml graduated cylinder for lipid separation.The chloroform/lipid layer of the filtrate was allowed to separate from the methanol/water layer for ∼10 min, and the methanol/water layer was removed via aspiration (Van Dyke et al., 2014).The remaining chloroform was evaporated using nitrogen gas.Total lipid was determined gravimetrically by dividing the mass of the extracted lipid by the mass of the subsample to calculate the proportion of the lipid relative to the total subsample mass.This was then multiplied by the total dry mass of the whole egg, giving the amount of lipid (g) in the entire egg.Total energy (kJ) was determined using a Parr 6200 calorimeter (Parr Instrument Company, Illinois, USA).A subset of each sample homogenate (∼0.4 mg) was pressed into pellets, weighed and combusted in the calorimeter (Thompson et al., 1999).The calorimeter was calibrated every 20 samples using benzoic acid.

Stable isotope analysis
Stable isotope analyses were used to validate the site differences in adult turtle diet found in Petrov et al. (2020) and determine whether the isotopic compositions (δ 13 C and δ 15 N) of the eggs reflected the isotopic compositions of their mothers.We analysed δ 13 C and δ 15 N of the claw clippings sampled from each gravid E. macquarii and δ 13 C and δ 15 N of the eggs.One milligram of claw homogenate and 1 mg of egg homogenate were weighed into tin capsules and packaged into 96-well microplates prior to analysis.δ 13 C and δ 15 N were determined using a Delta V Advantage isotope ratio mass spectrometer (Thermofisher Scientific, Waltham, MA, USA) coupled to a ConfloIV and FlashHT at the Centre for Carbon, Water and Food of the University of Sydney.For full isotope ratio mass spectrometer methodology, refer to Petrov et al. (2020).Isotopic values are presented in delta notation ( ), relative to Vienna Pee Dee Belemnite (VPDB) for carbon and ambient air for nitrogen.Precision was between 0.03 and 0.05 for carbon analysis (1 SD, n = 2) and between 0.02 and 0.04 for nitrogen analysis (1 SD, n = 2).

Statistical analyses Comparing isotopic composition of female E. macquarii across sites
We tested whether female E. macquarii differed in isotopic composition (δ 13 C and δ 15 N) among sites, to confirm that the adult turtle differences in isotopic composition reported in Petrov et al. (2020) were still present.We ran a multivariate analysis of covariance (MANCOVA) in SAS (PROC GLM) comparing the isotopic composition, δ 13 C and δ 15 N of E. macquarii females among sites.We set δ 13 C and δ 15 N as the dependent variables, site as the fixed main effect, maternal body size [straight carapace length (SCL)] as a covariate and the interaction of site and maternal body size as the effect of interest.

Effect of maternal diet on reproductive allocation
To determine the effect of maternal diet on reproductive allocation, we used wetland as a proxy for diet based on the among-wetland differences in diet we had previously reported (Petrov et al., 2020).We compared the following reproductive parameters across the four wetlands: total clutch mass (as an index of clutch size), individual egg mass and bulk egg composition (including water, protein, lipid, energy and isotopic composition).We also tested for among-site differences in hatching success to determine whether any egg composition differences we detected were related to differences in hatching success.To test for among-site differences in total clutch mass, we ran an analysis of covariance (ANCOVA) in SAS (PROC GLM) with log-transformed total clutch mass as the response variable, site as a fixed effect and log-transformed maternal body size (SCL) as a covariate.
To test for differences in individual egg mass, we ran an ANCOVA in SAS (PROC MIXED) with log-transformed egg wet mass as the response variable, site and laying order as fixed effects, log-transformed maternal body size as a covariate and maternal ID as a random effect.Laying order was included in the analysis as an additional covariate to test for potential egg size differences between eggs laid early in a clutch and those laid later (Loudon, 2014).Maternal ID was included as a random effect in the model to account for pseudoreplication of the clutches, because we included two eggs per clutch (Hurlbert, 1984).Maternal body size was included in the analysis of total clutch mass and individual egg mass because the volume of the mothers' body cavities and diameters of their pelvic openings may influence both clutch and egg width (Congdon, 1989).
To compare the moisture, protein, lipid and energy contents of each egg, we ran four separate ANCOVAs in SAS (PROC MIXED).The PROC MIXED procedure allows random variation to be modelled at both within-and betweensubject levels concurrently.For each separate analysis, we log-transformed moisture, protein, lipid and energy contents to meet the assumptions of an ANCOVA as evaluated by examination of residuals.Log-transformed moisture, protein,  and energy were set as the response variables in each analysis.In each analysis, site and laying order were included as fixed effects, log-transformed egg dry mass as a covariate and maternal ID as a random effect.To determine whether the isotopic compositions (δ 13 C and δ 15 N) of the eggs reflect the isotopic compositions of their mothers, we ran two separate ANCOVAs in SAS (PROC MIXED), one each for δ 13 C and δ 15 N. We set the isotopic composition of the eggs (δ 13 C or δ 15 N) as the response variable, the isotopic composition of the mothers (δ 13 C or δ 15 N) and site as fixed effects and maternal ID as a random effect.

Hatching success
To determine the effect of maternal diet on hatching success, we ran a generalized linear mixed model in SAS (PROC GLIMMIX).We set the response variable as the number of eggs that hatched divided by the number of eggs laid, with site and temperature as fixed effects and maternal ID as a random factor.Incubation temperature was included because it can affect embryonic physiology in reptiles (Deeming and Ferguson, 1991;Booth, 1998;Dormer et al., 2016).
In all ANCOVAs, we tested full factorial designs initially.When interactions were not statistically significant, their variance component was rolled into the error term.We report the full models in supplementary materials.Normality and homoscedasticity were assessed for all statistical tests by examining the plots of residuals graphically, in conjunction with the Shapiro-Wilk test (P > 0.05).All statistical tests were assessed at α = 0.05.Best-fit covariate structures for random effects were determined using Akaike's Information Criterion (AIC) in mixed models.Where there were more than two levels of a statistically significant main effect of interest, the significance was further examined using post hoc Tukey-Kramer multiple comparisons.

Results
Thirty-one gravid female E. macquarii were collected from the four wetlands (nine from Safes Lagoon, seven from Cockatoo Lagoon, eight from Longmore and seven from Gunbower Creek).

Isotopic composition of E. macquarii across sites
In the MANCOVA of maternal δ 15 N and maternal δ 13 C against site and maternal body size, no interaction terms were statistically significant (Supplementary Material, Table S1); their sums of squares were incorporated into the error term.Maternal δ 15 N and maternal δ 13 C differed significantly across sites (Supplementary Material, Table S2).Univariate analyses of maternal δ 15 N and maternal δ 13 C showed that the multivariate differences in site occurred for both maternal δ 15 N and maternal δ 13 C (Supplementary Material, Tables S3  and S4).Emydura macquarii from Cockatoo Lagoon were significantly lower in δ 13 C compared to E. macquarii from Gunbower Creek (P < 0.002), Longmore Lagoon (P < 0.001) and Safes Lagoon (P < 0.001; Supplementary Material, Table S5).Emydura macquarii from Safes Lagoon were significantly higher in δ 13 C compared to E. macquarii from Gunbower Creek (P < 0.003; Supplementary Material, Table S5).Emydura macquarii from Safes Lagoon had significantly lower δ 15 N compared to E. macquarii from Cockatoo Lagoon (P < 0.003), Gunbower Creek (P < 0.002) and Longmore Lagoon (P < 0.001; Supplementary Material, Table S5).Emydura macquarii from Longmore Lagoon had similar δ 15 N compared to Cockatoo Lagoon and Gunbower Creek (all P > 0.077).The pairwise among-site differences for Safes Lagoon, Cockatoo Lagoon and Gunbower Creek aligned with the site differences in adult turtle diet found by Petrov et al. (2018Petrov et al. ( , 2020) ) (Supplementary Material, Table S5 and Fig. S1), whereby E. macquarii from Safes Lagoon had a more herbivorous diet and Cockatoo Lagoon and Gunbower Creek exhibited an omnivorous diet.While E. macquarii from Longmore Lagoon were still high in δ 15 N in this study (Supplementary Material, Table S5), the difference was no longer significant, as reported in 2020.

Effect of maternal diet on reproductive allocation Total clutch mass
The interaction between site and maternal body size on total clutch mass was significant (P < 0.038; Table 1).Average total clutch mass was 177.1 ± 41.04 g (range, 119.3-250.1 g) at Cockatoo Lagoon, 189.7 ± 42.0 g (range, 137.9-250.8g) at Gunbower Creek, 191.0 ± 23.5 g (range, 159.9-225.4g) at Longmore Lagoon and 180.0 ± 43.3 g (range, 107.5-217.8g) at Safes Lagoon (Supplementary Material, Tables S6-S9).There was a positive relationship between total clutch mass and maternal body size at each site (Fig. 1; Supplementary Material, Table S10).At Longmore Lagoon, the slope of the relationship between maternal body size and total clutch mass is significantly smaller than at the other three sites (Fig. 1; Supplementary Material, Table S10).Thus, clutch size of Longmore turtles increased at a slower rate relative to body size than it did in turtles from the other three sites (Fig. 1).Also, at all body sizes, E. macquarii at Cockatoo Lagoon produced significantly smaller clutches in   comparison to E. macquarii at Safes Lagoon and Gunbower Creek, though the magnitude of the difference was slight (Fig. 1; Supplementary Material, Table S10).

Egg mass
Individual egg mass did not differ across sites (P = 0.099; Table 2).Individual egg mass was influenced by an interaction between laying order and maternal body size (SCL, P < 0.010).The interactions of order and site and order, maternal body size and site were borderline (all P > 0.050), and there were no other significant interactions (Table 2).
To determine how egg size is affected by the interaction of laying order and maternal body size, we examined the slopes of the linear relationship between egg mass, laying order and maternal body size (Table 3).Smaller turtles tended to produce larger eggs later in a clutch, whereas larger turtles tended to produce larger eggs earlier in a clutch.The interaction between laying order and maternal SCL is statistically significant, but the effect is small (Table 3).

Egg composition
In the four ANCOVAs of egg moisture, protein, lipid and energy compared across site, laying order, egg dry mass and maternal ID, no interactions were statistically significant (Supplementary Material, Tables S11-S14); their sums of squares were incorporated into the error term.The moisture content (P < 0.001), protein content (P < 0.001), lipid content (P = 0.001) and energy content (P < 0.001) of the eggs differed significantly with egg dry mass but did not differ significantly with site (all P > 0.154) or laying order (all P > 0.160; Supplementary Material, Tables S15-S18).Thus, egg compositions did not differ across sites in this study.
Since there were no differences in egg composition in our study, the average egg wet mass was 9.98 ± 0.16 g, and the average water fraction was 78.41 ± 0.30%.After removing water, the average egg lipid content was 14.52 ± 0.64% dry mass, and the average protein content was 43.42 ± 0.72% dry mass.The average energy content was 23.29 ± 0.21 kJ/mg dry mass.More compositional details are available in the Supplementary Material (Tables S6-S9  "Egg δ 15 N was influenced by maternal δ 15 N (P < 0.001); however, there were no other significant interactions (Table 4).To determine how egg δ 15 N is affected by maternal δ 15 N, we examined the slope of the linear relationship between egg δ 15 N and maternal δ 15 N, which revealed a significant positive relationship between egg δ 15 N and maternal δ 15 N (Fig. 2a, Table 5).The slope of the relationship between egg δ 15 N and maternal δ 15 N was very close to one, and the yintercept was approximately 1 (Fig. 2a, Table 5), suggesting mothers allocate slightly more 15 N than 14 N to their eggs, but at a rate that is not influenced by their own δ 15 N. Similarly, egg δ 13 C was influenced by maternal δ 13 C (P < 0.001); however, there were no other significant interactions (Table 4).To determine how egg δ 13 C is affected by maternal δ 13 C, we examined the slope of the linear relationship between egg δ 13 C and maternal δ 13 C, which revealed a significant positive relationship between egg δ 13 C and maternal δ 13 C (Fig. 2b and Table 5).The slope of the relationship between egg δ 13 C and maternal δ 13 C was just slightly less than 1, and the y-intercept was not different from zero (Fig. 2b; Table 5), suggesting mothers are allocating similar ratios of heavy and light carbon into their eggs as they themselves have.The very slight depletion in the slope suggests that at most, mothers are allocating slightly less carbon 13 C to their eggs than carbon 12 C.

Discussion
Maternal diet influences how energy and nutrients are allocated to offspring during reproduction.We found a positive relationship between clutch mass and maternal body size in turtles from each site, with clutch mass increasing with maternal body size.This finding was expected, as an increase in clutch mass with increasing maternal body size is common across oviparous species (Gatto et al., 2020).At Longmore Lagoon, where food is limited (Petrov et al., 2018), clutch size increased at a slower rate relative to body size than at the other three sites, suggesting a potential constraint of maternal diet on reproductive allocation.Our previous studies found female E. macquarii at Longmore Lagoon tend towards carnivory and have higher rates of empty stomachs and lower body condition (Petrov et al., 2018;Petrov et al., 2020).Although our analysis found that δ 15 N of E. macquarii from Longmore Lagoon in this study was no longer significantly different to δ 15 N of E. macquarii from the intermediate sites of Cockatoo Lagoon or Gunbower Creek, individual E. macquarii from Longmore Lagoon tended to have the highest values in the study, suggesting some individuals may be trending towards carnivory, as previously reported.In general, there may be greater herbivory occurring now at Longmore than we observed in the prior study.We previously suggested that the scarcity of food at Longmore Lagoon drove the reduced body condition we detected in E. macquarii (Petrov et al., 2020).Here, our data further suggest that reduced food abundance also drives reductions in the number of eggs that a female can produce.Future studies should experimentally manipulate the amount of food (especially filamentous green algae) available to E. macquarii to confirm the correlations we have observed between algae availability and the number of eggs a female produces.Likewise, laboratory-based studies that identify how physiological allocation decisions (Dunham et al., 1989) change, which result in the patterns we report here, would be excellent in order to fully understand the mechanisms underlying the shifts in fecundity that we observed.
Our results are similar to those previously reported in other reptiles.For example, adult female brown anole lizards (Anolis sagrei) raised under low prey availability produced significantly fewer eggs per clutch than females raised under high prey availability (Warner et al., 2015).A similar trend has also been observed in reproductive female phrynosomatid lizards (Sceloporus virgatus), with females producing smaller clutch sizes when prey availability and rainfall are low (Abell, 1999).Clutch size in turtles is likely determined by maternal body condition and the availability of prey in late summer and autumn, as follicles begin to enlarge and continue to mature through to winter (Harless and Morlock, 1979;Georges, 1983;Wilkinson and Gibbons, 2005).As such, the availability of prey at this time likely influences the amount of energy available for reproduction.E. macquarii at Safes Lagoon and Gunbower Creek.Emydura macquarii at Cockatoo Lagoon have similar diets to E. macquarii at Gunbower Creek (Petrov et al., 2020), with both sites having similar levels of turbidity and filamentous green algae abundance (Petrov et al., 2018).Because of the similarities between the environment and diets of E. macquarii at Cockatoo Lagoon and Gunbower Creek, it was expected that any effect of maternal diet observed at one of these sites would likely be present at the other.However, no reduction in clutch size at Gunbower Creek in comparison with Cockatoo Lagoon was detected.The smaller clutch sizes observed at Cockatoo Lagoon may be among-year variations in maternal diet that our stable isotope approach was not able to detect.Environmental sampling and isotopic sampling by Petrov et al. (2018 and2020) were conducted in 2015 and the start of 2016, while the reproductive allocation data in the present study were collected at the end of 2016.If prey availability or abundance at Cockatoo Lagoon declined early in 2016, at the time when follicles begin to enlarge (Harless and Morlock, 1979;Georges, 1983), E. macquarii females may have had less energy available for reproduction than E. macquarii at Gunbower Creek.Differences in prey availability or abundance could have also occurred at the other sites; however, this was not detected through the reproductive allocation data.As such, future studies examining reproductive allocation should sample the environment and diet (i.e.stomach content and isotopic sampling) throughout reproduction.
We found no significant effect of maternal diet on egg mass, with female E. macquarii producing similar-sized eggs across sites.This result suggests that females alter the number  of eggs produced in response to prey availability but do not alter egg size.This result is similar to the findings of Warner et al. (2015) who found that female brown anole lizards A. sagrei raised on a low prey diet produced similarsized eggs to females on a high prey diet.Egg size in the current study did however vary with an interaction of laying order and maternal body size (Congdon and Gibbons, 1985;Congdon and Gibbons, 1987).Freshwater turtles construct nest chambers shaped like a flask, with the bottom of the nest being wider in diameter than the top (Goode, 1965).The wider base allows more eggs to be incubated at the bottom of the nest (Booth, 2010), away from daily temperature fluctuations (Thompson, 1988).Smaller turtles may produce smaller eggs earlier in a clutch to ensure more eggs are protected against temperature fluctuations.Laying bigger eggs at the bottom of a nest has also been hypothesized to support the mass of the eggs at the top of the nest during development (Tucker and Janzen, 1998); however, this hypothesis and reasons for bigger eggs being laid later in a clutch are yet to be explored.
We found no effect of site on the compositions of eggs (i.e.moisture, protein, lipid or energy), suggesting E. macquarii at different sites are allocating similar proportions of nutrients into their eggs regardless of maternal diet.However, this study does not take into account the proportions of inorganic ions such as calcium, sodium, magnesium or iron, which constitute 5% of an eggs composition (Thompson et al., 2000), or the proportions of specific amino or fatty acids (Speake and Thompson, 1999;Thompson et al., 2000).Elevated levels of omega-3 fatty acids in particular increased cognitive maturation in ring-billed gulls (Larus delawarensis), which led to earlier fledging (Lamarre et al., 2021).Further research should examine the proportion of these additional macro-and micronutrients in eggs to determine whether maternal diet is influencing egg composition and potentially hatchling size and physiology at a scale not studied here.
Female turtles allocated similar ratios of maternal δ 15 N and maternal δ 13 C to their eggs as they had available in their bodies.Offspring are expected to have δ 15 N values that are approximately one trophic position higher than their mother, as offspring are consuming resources that are derived from their mother (Jenkins et al., 2001), and some of this difference may occur at the maternal allocation step.Our analysis found that females were allocating slightly more 15 N to their eggs, enough to increase the δ 15 N by about 1.This is less than a full trophic level increase (Post, 2002) but still indicates a slight enrichment.Females were also found to allocate similar ratios of heavy and light carbon into their eggs, as found in their own bodies.
The correlation observed between maternal δ 15 N and δ 13 C and egg isotopic δ 15 N and δ 13 C was similar to those found in other studies of turtles (Caut et al., 2008;Zbinden et al., 2011;Frankel et al., 2012;Carpentier et al., 2015).For instance, in leatherback turtles (Dermochelys coriacea), a positive relationship was found between maternal blood δ 15 N and δ 13 C and egg yolk δ 15 N and δ 13 C (Caut et al., 2008), while in loggerhead turtles (Caretta caretta), a significant relationship was found between egg yolk δ 15 N and δ 13 C and respective adult tissues (Carpentier et al., 2015).Whole blood of C. caretta was the best predictor for egg yolk isotopic values, with loggerhead turtle egg yolk enriching in δ 15 N by ∼1 trophic position (Carpentier et al., 2015), as observed in the current study.A distinguishing difference between our study and those mentioned above is the samples used.In the current study, we analysed δ 15 N and δ 13 C from whole turtle eggs and maternal claws, whereas other studies have isolated egg yolk and compared this to maternal blood (Caut et al., 2008;Carpentier et al., 2015) and maternal carapace (Zbinden et al., 2011).Despite these sampling differences, we observed similar relationships between egg isotopic values and maternal isotopic values.

Figure 1 :
Figure 1: Relationships between total clutch mass and maternal body size, compared across the algae-rich site, Safes Lagoon; algae-poor site, Longmore Lagoon; and intermediate sites, Cockatoo Lagoon and Gunbower Creek.

Table 1 :
ANCOVA results comparing the relationships between total clutch mass, across sites and log-transformed maternal body size (SCL)

Table 2 :
ANCOVA comparing the relationships between individual egg mass, across sites, order and log-transformed maternal body size (SCL)

Table 3 :
Parameter estimates (±SE) of the relationships between individual egg mass, laying order and log-transformed maternal body size (SCL)

Table 4 :
ANCOVA comparing the relationship between the isotopic composition of the eggs (δ 15 N and δ 13 C) with the isotopic composition of the mothers (δ 15 N and δ 13 C), across sites

Table 5 :
Parameter estimates (±SE) for the relationships between (i) egg δ 15 N and maternal δ 15 N, and (ii) egg δ 13 C and maternal δ 13 C and associated P values